Basic Illuminating Light Paths and Proper Microscope Alignment

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Basic Illuminating Light Paths and Proper
Microscope Alignment

• E. D. Salmon
• University of North Carolina at Chapel Hill

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Today

• Answers to last lectures questions
• Light Paths for Trans illumination
• Koehler Illumination
• Action of Field and Condenser Diaphragms
• Conjugate Planes in Properly Aligned Microscope
• Major components of Research Microscope

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Homework
A beam of light in glass hits a surface at an
angle. At what angle does the light just become
total internally reflected if the glass has a
refractive Index of 1.52 and the interface has a
refractive index of a. Air b. Water c.
Immersion oil In each case, what is the numerical
aperture (NA) of the beam relative to the normal
to the interface?
1.
q1
90
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Homework 2 What is an easy way to measure the
approximate focal length of a lens
LAMP
f
LAMP
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Homework 3 What is The Ocular Focal Length for
the Following Magnifications?

• 5X _________
• 10X _________
• 20X _________
• 25X _________

foc 255/Mag (mm)
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Homework 4 For Finite Focal Length Objective and
OTL 160 mm, what is focal length for the
following Objective Magnifications

• 4X _________
• 10X ________
• 20X ________
• 40X ________
• 60X ________
• 100X _______

(mm)
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A Lamp Collector Lens and Microscope Condenser
Lens are Used to Concentrate Light on the Specimen
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Two Schemes for Specimen Illumination Using a
Condenser
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Critical Illumination
Light source out-of-focus at condenser aperture
and in-focus at specimen. Produces bright, but
un-even illumination of specimen.
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Koehler Illumination
Light source in-focus at condenser aperture and
out-of-focus at specimen. Produces bright, and
even illumination of specimen.
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The Field Diaphragm Controls the Area Illuminated
The field diaphragm is focused onto the specimen
by moving the condenser back-and-forth The lamp
image is focused at the condenser aperture
(diaphragm) by moving the Collector lens
Back-and forth.
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The Condenser Diaphragm Controls the Illumination
NA
An image of the Condenser Diaphragm is in-focus
in the Objective Back Focal Plan (Aperture). As
the condenser diaphragm is opened, the
illumination NA increases without changing the
area of specimen Illuminated (area controlled by
Field Diaphragm).
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Summary of Koehler Illumination

• The lamp filament and collector lens must both be
  centered on the same optical axis in the lamp
  housing.
• The collector lens is used to project an image of
  the lamp centered and in-focus at the condenser
  diaphragm.
• The condenser lens is used to project an image of
  the field diaphragm centered and in-focus on the
  specimen.

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Summary of Koehler Illumination (cont.)

• A telescope is used to view the objective back
  back focal plane (aperture) in order to
• Adjust the opening of the condenser diaphragm so
  that the diameter of its image is equal to or
  slightly less than the diameter of the objective
  aperture
• Adjust the focus and x-y position of the lamp
  image so it is centered and in-focus at the
  objective back aperture

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Collector Lens in Lamp Housing is Translated
Along Optical Axis to Bring Lamp Image into Focus
at Condenser Front focal Plane (Diaphragm)
Condenser focus
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Condenser is Translated Along Optical Axis to
Bring Field Diaphragm into Focus
Condenser X-Y Translation Screws Are Used
to Center Image of Field-Diaphragm
Condenser focus
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Image Planes for The Field Diaphragm And Light
Source (Condenser Diaphragm) Alternate
Along Optical Path of a Properly Aligned
Microscope
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The Light Path is Extended or Re-Directed Using
Projection Lenses, Mirrors and Prisms and
Beam-Switches
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(No Transcript)
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Homework Problem 5
  The light source is a 3-mm square tungsten
filament. The design of the illumination system
requires that (1) the filament be 300 mm away
from the condenser diaphragm, (2) the image of
the filament must be in focus at the condenser
diaphragm and (3) the filament must be 15-mm
square to fill the condenser aperture with
light. Assuming the lamp collector lens is an
ideal thin lens, determine the focal length, and
the position of the collector lens between the
lamp filament and the condenser diaphragm.
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Homework Problem 6
A field diaphragm or iris is placed in front of
the collector lens as shown for the Koehler
illumination system. The field iris is used to
control the illuminated area of the specimen.
The condenser lens is translated back and forth
along the central axis until an image of the
field diaphragm is in sharp focus on the
specimen. When the opening of the field
diaphragm is 20 mm, the image on the specimen
must be 2 mm in diameter. In addition, the
field diaphragm is placed 160 mm away from the
condenser lens. What is the focal length of the
condenser needed to meet these requirements?
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Homework Problem 7
Indicate In-focus or out-of-focusfor Fiel
d Diaphragm Light Source at Field
Diaphragm _____________ ____________ Condenser
Diaphragm _____________ ____________ Specimen __
___________ ____________ Objective BFP
_____________ ____________ Ocular
FFP _____________ ____________ Ocular BFP
(RamdensDisk) _____________ ____________ Retina
(or camera detector) _____________ ____________
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Homework 8
Work through the Microscope Illumination Section
under Microscope Anatomy at http//micro.magnet
.fsu.edu/primer/index.html
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Homework Problem 9 Identify Major
Components And Their Locations And
Functions Within Modern Research
Light Microscope (See Salmon And Canman, 2000,
Current Protocols in Cell Biology, 4.1)
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Condenser NA Relative to Objective NA
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A Teloscope is Often Used to Observe the Image of
the Lamp and Condenser Diaphragm at the Objective
Back focal Plane
-Remove Ocular and insert Teloscope to view
objective back aperture (back focal plane) -In
some microscopes, a focusable Bertrand lens is
inserted between the ocular and objective to
produce a teloscope while the ocular